Combined effects of nitrate and medium-chain fatty acids on methane production, rumen fermentation, and rumen bacterial populations in vitro

This study investigated the combined effects of nitrate (NT) and medium-chain fatty acids (MCFA), including C8, C10, C12, and C14, on methane (CH4) production, rumen fermentation characteristics, and rumen bacteria using a 24 h batch incubation technique. Four types of treatments were used: control (no nitrate, no MCFA), NT (nitrate at 3.65 mM), NT + MCFA (nitrate at 3.65 mM + one of the four MCFA at 500 mg/L), and NT + MCFA/MCFA (nitrate at 3.65 mM + a binary combination of MCFA at 250 and 250 mg/L). All treatments decreased (P < 0.001) methanogenesis (mL/g dry matter incubated) compared with the control, but their efficiency was dependent on the MCFA type. The most efficient CH4 inhibitor was the NT + C10 treatment (− 40%). The combinations containing C10 and C12 had the greatest effect on bacterial alpha and beta diversity and relative microbial abundance (P < 0.001). Next-generation sequencing showed that the family Succinivibrionaceae was favored in treatments with the greatest CH4 inhibition at the expense of Prevotella and Ruminococcaceae. Furthermore, the relative abundance of Archaea decreased (P < 0.05) in the NT + C10 and NT + C10/C12 treatments. These results confirm that the combination of NT with MCFA (C10 and C12 in particular) may effectively reduce CH4 production.

NT mainly functions as an electron sink because NT reduction is thermodynamically more favorable than CO 2 reduction 10 .Additionally, NT is toxic to protozoa 10 , and its intermediate nitrite is toxic to methanogens 26,27 .MCFA are antibacterial and antiprotozoal agents 19,28,29 as they dissociate in bacterial cells 17 , may cause defaunation 30,31 , and, therefore, lower H 2 supply 5,9 .Additionally, MCFA can directly inhibit methanogens 29,32 .The target microorganisms of NT and MCFA appear to overlap, but their effects may be complementary.Mixing low doses of NT and MCFA (and their binary combinations) may decrease rumen methane production without inhibiting fermentation.
Our hypotheses were as follows: (1) different combinations of MCFA and NT would vary in their effectiveness in mitigating CH 4 production, and (2) efficient combinations of MCFA and NT capable of lowering methanogenesis without negatively affecting rumen fermentation may be found.
The nVFA (P = 0.031) and the acetate: propionate ratio (P = 0.065) were not altered by any treatment.The effects of NT + MCFAs on VFA production and individual proportions are shown in Table 2. NT + C10 significantly decreased (P < 0.05) the molar proportion of acetate and increased (P < 0.05) the molar proportions of butyrate, valerate, and iso-valerate.Furthermore, butyrate proportions were significantly reduced (P < 0.05) with the following treatments: NT, NT + C14, and NT + C12/C14.Finally, only the NT + C8 and NT + C10/C12 treatments substantially decreased (P < 0.05) the molar proportion of valerate compared to the control.
The pH increased (P < 0.05) in all treatments relative to the control; the highest increase was recorded for the NT + C10.NH 3 -N concentrations increased (P < 0.05) in all NT + MCFA treatments, with NT + C10 increasing NH 3 -N concentrations the most.

Table 1.
Effects of MCFA + NT on in vitro gas and methane production, apparent dry matter disappearance (aDMd), ammonia-N concentration, and pH.† Total gas production; ‡ apparent dry matter disappearance; § dry matter incubated; ¶net production of volatile fatty acids.*Means within a column differ significantly (P < 0.05) from the corresponding control (0 mg/L).SEM, standard error of the mean.

Microbial community
Bacterial diversity varied greatly among treatments and was expressed as ASV counts (Fig. 1) and indices of diversity (Table 3).All indices of alpha diversity significantly (P < 0.05) decreased when the treatments contained C10 or C12, with the NT + C10 treatment having the strongest effect on microbial diversity, richness, and evenness (Figs. 1, 4, Table 3).In contrast, the smallest effect on diversity was observed for treatments containing C8 and C14.Additionally, the NT + C10 and NT + C10/C12 treatments significantly decreased (P < 0.05) the relative abundance of Archaea (Table 4).
The most abundant phyla (Fig. 2a) were Proteobacteria (relative abundance range 19.5-68.4%),followed by Firmicutes (30.2-43.8%),and Bacteroidota (0.96-37.98%).The dominance of the phylum Proteobacteria was not consistent across all treatments, and the dominance of the respective phyla varied based on the type of MCFA.Proteobacteria thrived in the C10 treatment group at the expense of Bacteroidota.The phylum Bacteroidota showed signs of particular sensitivity to MCFA, as its relative abundance was highest in the NT treatment.Firmicutes, conversely, appear to be favored in treatments containing C8.To gain further insight into the bacterial community, the relative microbial abundances are also shown at the class (Fig. 2b), family (Fig. 2c), and genus (Fig. 2d) levels.The highest relative abundance at the family level was noted for Succinivibrionaceae (25.2-70.7%),followed by Prevotellaceae (0.5-34.5%) and Lachnospiraceae (14.9-23.2%).In general, the family Succinivibrionaceae showed an increasing trend in treatments containing C10 and/or C12.In contrast, the C10 treatment simultaneously decreased the relative abundance of Prevotellaceae.
Overall, compared with the control, the treatments containing C10 and C12 had the strongest effect on the relative abundances of bacteria and Archaea.The weakest antimicrobial activity was observed in the C8 and C14 treatments and their respective combinations.Compared to the combination with MCFA, NT alone had little effect on the composition of the microbiota in this experiment.This was confirmed by analysis of Compositions of Microbiomes with Bias Correction (ANCOM-BC) implemented at a family level (Fig. 4) and nonmetric multidimensional scaling (NMDS) analysis used to visualize and assess community composition (Fig. 3).
In the NMDS analysis, the treatments exhibited distinct clustering patterns on the plot.Specifically, treatments comprising control, NT, C8, and C14 formed a cluster located in the upper-left quadrant of the plot (Fig. 3), indicating a high degree of similarity in their microbiome compositions.Conversely, treatments involving C10 and C12 displayed a tendency to be positioned towards the right side of the plot, suggesting differences in microbiome composition compared to the former group.

Discussion
By combining low doses of NT and MCFA, we confirmed our hypotheses that (1) individual combinations varied in their CH 4 mitigating efficacy and (2) there was one superior treatment (NT + C10) that decreased methanogenesis the most without negative effects on fermentation.In addition, the microbial community shifted to resemble the rumen microbiota of low-CH 4 emitting cattle, with low Ruminococcaceae 33,34 (e.g., 4.3% 33 ), and high Succinivibrionaceae 35 (e.g., 8.9-9.3% 36,37).Combinations of NT and various MCFA differed in their CH 4 suppressing effect, confirming previous findings that CH 4 mitigation is dependent on the MCFA type 19 .Overall, our results suggested that the most effective combinations were those containing C10.The most effective treatment was NT + C10 (− 40%; mL/g DMi).This is consistent with the anti-methanogenic effect of C10 reported by Goel et al. (2009), who showed a dose-dependent effect of C10 17 .In their study, C10 at 400 and 600 mg/L of incubation medium decreased CH 4 production by 44% and 88%, respectively, but also inhibited overall fermentation, as suggested by decreased VFA production 17 .In contrast, Dohme et al. 18 reported no effect of C10 (approximately 600 mg/L) on methanogenesis using a RUSITEC, an in vitro rumen simulation technique.Previous in vitro studies suggested that CH 4 is reduced by about 5% for each mmol of NT 38,39 .However, in our study, it was only about 3.3% per mmol of NT, indicating an incomplete reduction of NT.Latham et al. (2016)  40 previously reported that the mitigation potential of NT may be reduced due to its partial metabolization to nitrous oxide either via dissimilatory NT reduction or via incomplete denitrification.
The most efficient binary combination of MCFA in the present study was NT + C10/C12.When expressed as CH 4 production per VFA, this treatment inhibited CH 4 production to the greatest extent.Consistent with this, Desbois and Smith 29 reported that MCFA with 10 or 12 carbons is the most biologically active and that the antimicrobial activity of MCFA decreases with any change in carbon chain length.C12 is one of the most frequently examined MCFAs 19 .In previous studies, C12 has decreased CH 4 production in the rumen by up to 89% in vitro 18,41 and by as much as 76% in vivo 42,43 .
The fact that binary combinations of MCFA might be effective anti-methanogenic additives was demonstrated by Soliva et al. 36 In their study, C12 and C14 exhibited synergistic CH 4 suppression effects.Their various proportions decreased methanogenesis by 50-96%, and the extent of inhibition increased with increasing amounts of C12 in the mixture.However, in our study, NT + C12/C14 decreased methanogenesis by much less in comparison with the study of Soliva et al. 36 The lower efficiency of our binary combination may be attributed to the higher concentration used in their study (1000 mg/L vs. 500 mg/L in our study).Another reason could be the C12/ C14 ratio.CH 4 production was not as inhibited in our study as it was in the study of Soliva et al. 36 , where CH 4 production progressively decreased with an increasing proportion of C12 in the mixture.We assessed a 1:1 ratio, whereas the most effective ratio reported by Soliva et al. 36 was 2:1 or higher.
In general, the treatments that did not contain C10 or C12 were less effective.Namely, NT with C8, C14, and their binary combination decreased methanogenesis only by 16-24%.In line with this, C14 has been reported to have low efficiency 14,18,20 , and the low potential for CH 4 inhibition by C8 is in agreement with the findings of Ajisaka et al. 44 .
In general, individual MCFA with NT had little effect on nVFA production or individual VFA proportions.This is in line with the findings of Yanza et al. (2020), who showed that VFA concentrations were not significantly decreased by MCFA in vitro but only in vivo.Furthermore, in our study, sole NT treatment had no effect on nVFA, supporting the results of other in vitro studies with NT 38,45 .The P-value in the analysis of variance was statistically significant (P = 0.031); however, Dunnett´s post-hoc analysis failed to identify significant differences between the treatments (P > 0.05).Nevertheless, the numerical differences showed the NT + C10 treatment decreased nVFA the most (− 14.2%).Previously, similar doses of C10 (400-600 mg/L) decreased total VFA production by 17-23% 17,18 .This difference could be due to the NT in our treatments, which might have mitigated the inhibitory effects of MCFA on nVFA production, as NT has previously increased VFA concentrations 46,47 .Conversely, treatments C8 and C14 had the lowest effect on rumen fermentation, as indicated by their weak effect on CH 4 inhibition.In line with the generally low inhibitory effect on CH 4 production, the effects of the C8 and C14 treatments on nVFA production were negligible.
The molar proportions of the individual VFA were most prominently affected by the NT + C10 treatment.These effects are consistent with the strong anti-methanogenic and antimicrobial activities of C10 29 .The NT + C10 treatment decreased acetate production.Acetate formation, an H 2 releasing pathway, usually decreases at higher H 2 concentrations, which occurs when methanogenesis is inhibited 48,49 .High H 2 concentrations favor H 2 sinks such as propionate, butyrate, and valerate [48][49][50] .We did not measure the levels of H 2 , however, this theory was confirmed by an increase in butyrate concentrations, which, as demonstrated in a fermentation balance experiment, may provide 14% of the H 2 sinks in the rumen 51 .Propionate is a more common H 2 sink than butyrate 51 ; however, its concentration did not increase.This could be because propionate-producing bacteria were inhibited.Indeed, the propionate-producing families Prevotellaceae, Veillonellaceae, and Selenomonadaceae 52,53 were reduced, but at the expense of Succinivibrionaceae, which produce succinate, a precursor to propionate 52 .As a result, another explanation could be that the bacteria producing propionate from succinate were inhibited.Furthermore, the added NT, a favorable H 2 sink 49 , was available and could have consumed the free H 2 required for propionate production 54 .Acetate is normally produced by cellulolytic microorganisms along with H 2 50,55 .The decrease in acetate might be due to C10 inhibiting cellulolytic microorganisms, such as the family Ruminococcaeae 56 , which was inhibited in the NT + C10 treatment along with acetate production.
The effects of MCFA on digestibility are type-and dose-dependent 19,31,57 .Higher doses of MCFA typically decrease nutrient digestibility both in vivo and in vitro 19,31,57 .For example, in in vitro continuous culture, C8, C10, and C12 at 5% DM (approximately 600 mg/L) reduced NDF digestion by 2.4, 6.0, and 8.7%, respectively 18 .This reduction may be due to the absorption of fatty acids (FA) on feed particles, limiting the access of enzymes and microbes, and/or FA may be directly absorbed by fiber-degrading microbes (protozoa or cellulolytic bacteria), to which they are toxic 19,24 .
Nevertheless, in the present study, digestibility was not influenced by MCFA, presumably because of their low dosages.The only exception was the NT + C14 treatment (− 6.7%), which had no significant effect on rumen fermentation or nVFA, and the proportion of microorganisms was very similar to that of the control.This result is similar to that of Dohme et al. (2001), who reported the lowest organic matter degradability in C14 (− 7.4%).NT alone (3.65 mM) did not decrease digestibility in our study.Previously, 5 mM NT did not decrease digestibility as well 27 .However, higher doses of NT may be toxic to cellulolytic bacteria and decrease digestibility in vitro 27 .
All treatments, including NT alone, increased pH in our study.This increase may have been due to the addition of NT, which is reduced to ammonia in the rumen.These results are consistent with those of Zhou et al. 26 , who reported an increase in pH at higher NT concentrations.In our study, the NT + C10 treatment increased the pH to a maximum of 6.27, which is in line with the numerically lower nVFA levels in this treatment.Similarly, Dohme et al. 18 reported the highest pH when supplemented with C10.However, all values of ruminal pH remained within the physiological range (5.5-7.5 54,55 ).
Various combinations of MCFA with NT added to ruminal fluid significantly affected the richness and diversity of bacterial populations to different degrees (Table 3, Fig. 1).Currently, treatments containing C10 and/or C12 significantly decreased the alpha diversity indices (Table 3), consistent with their effects on other ruminal parameters in our study and their strong antibacterial activity reported previously 17,18,29 .Burdick et al. 31 used a mixture of MCFA (C8, C10, and C12) and, contrary to our results, did not report any changes in alpha diversity.This might be due to the low concentrations used in their study, although there was a reported tendency to reduce the bacterial richness.Previous studies on MCFA either did not investigate ruminal microbiota 17,32 or used less sophisticated (chamber counting method) and insufficiently specific methods 18 .
The dominant bacterial phyla in the current study were Firmicutes, Bacteroidota, and Proteobacteria; this is consistent with the findings of previous studies 47,52,58 .The treatments (particularly those containing C10 and C12) that decreased CH 4 production also increased the relative abundance of Proteobacteria (Fig. 2a).Proteobacteria predominantly belonged to the family Succinivibrionaceae (Fig. 2c).Hydrogen plays a central role in CH 4 production 59,60 .The amount of H 2 in the rumen can be influenced by the abundance of H 2 -producing and H 2 -consuming bacteria associated with CH 4 emissions 61 .Previously, low-CH 4 -emitting ruminants and tammar wallabies were associated with the H 2 -consuming family Succinivibrionaceae 34,35,62 .Succinivibrionaceae utilize H 2 to generate succinate (a precursor to propionate) and, therefore, can reduce CH 4 emissions 52 .Treatment with NT + C10 increased the relative abundance of Succinivibrionaceae the most (70.7%) (Fig. 2c).In contrast, Vol:.( 1234567890 www.nature.com/scientificreports/Succinivibrionaceae were the least abundant in the NT + C14 treatment (25.2%).NT + C10 and NT + C14 treatments decreased CH 4 production the most and least, respectively.Within the phylum Bacteroidota, treatments with greater inhibition of methanogenesis decreased the relative abundance of the genus Prevotella (Fig. 2d).The genus Prevotella utilizes H 2 and produces propionate 61,63,64 ; in a study with Colombian buffalos, a higher abundance of Prevotella was associated with lower CH 4 emissions 65 .However, our findings are not consistent with this, as the genus Prevotella was less abundant in treatments resulting in lower CH 4 production.This lower relative abundance of Prevotella could be explained by H 2 availability in our study.Theoretically, the low H 2 availability may have been due to the H 2 being used for the reduction of supplemented NT to ammonia 63,66 .Furthermore, H 2 could have been utilized by the family Succinivibrionaceae, and therefore outcompeted the Prevotella genus.This shift in relative abundance from Prevotella to Succinivibrionaceae has been previously noted 67,68 .Furthermore, the relative abundance of Prevotella is decreased by supplementation with NT 69 .However, the majority of the previous studies on NT have reported an increase in the relative abundance of Prevotella 12,64,70,71 , because Prevotella is associated with nitrate metabolism 71 .
In the phylum Firmicutes, treatments with the greatest anti-methanogenic potential (NT + C10 and NT + C10/ C12) decreased the relative abundance of the family Ruminococcaceae (Fig. 2c).The Ruminococcaeae belong to the H 2 -producing bacteria 72 and are present in higher abundance in high CH 4 -emitting rumens 33,34 .This finding is consistent with our However, this family plays a significant role in fiber metabolism, and its reduction may cause a decrease in fiber digestion 73 .Unfortunately, we did not measure fiber digestion, and aDMd was not negatively affected.
A decrease in methanogenesis was also observed in the methanogenic population.The relative abundance of Archaea, the sole producers of CH 4 in the rumen 60 , was decreased by NT + C10 and NT + C10/C12 treatments by 84.2% and 45.7%, respectively (Table 4).Dohme et al. 18 and Burdick et al. 31 reported no change in the methanogen population when supplemented with MCFA (C8, C10, and C12).A meta-analysis showed that the Archaea population diminished quadratically only under in vitro conditions with increasing doses of MCFA 19 .Furthermore, NT supplementation decreases methanogenesis and consistently reduces the abundance of methanogenic Archaea 60,69 .Notably, it has been reported that instead of the overall abundance of methanogens, the community structure of methanogens 60 and differential gene expression of methanogenesis pathways are the decisive factors in ruminal methanogenesis 74 .

Conclusion
This in vitro study showed that the effects of NT and MCFA combinations depend on the type of MCFA.The tested treatments have the potential to lower CH 4 production without negatively affecting ruminal fermentation.The most effective CH 4 inhibitors were combinations of NT and C10/C12.These combinations also had the greatest impact on the ruminal microbiota, as reflected in the changes in bacterial diversity and shifts in the relative abundances of bacteria and Archaea.The reported results from our 24 h batch incubations should be verified for their long-term effects in vitro and efficiency in vivo.

Ethical compliance
Procedures with animals were conducted in accordance with Czech legislation (Act No. 246/1992 Coll., on the protection of animals against cruelty) and applicable European guidelines and regulations (Directive 2010/63/ EU, on the protection of animals used for scientific purposes) for experimentation with animals.The experimental protocol was approved by the Animal Ethics Committee of the Institute of Animal Science (Prague, Czech Republic).This study was conducted in accordance with ARRIVE guidelines to ensure an appropriate animal care.The donor cows were housed at the experimental farm of the Institute of Animal Science (Netluky, Prague, Czech Republic).

Treatments and experimental design
A 24 h batch incubation was used to evaluate the combined effect of NT and MCFA on CH 4 production and rumen fermentation.The treatments were: control (no NT, no MCFA); NT (nitrate at 3.65 mM), NT + MCFA (nitrate at 3.65 mM + one of four MCFA (C8, C10, C12, and C14) at 500 mg/L), and NT + MCFA/MCFA (nitrate at 3.65 mM + binary combination of MCFA (C8/C10, C8/C12, C8/C14, C10/C12, C10/C14, and C12/C14) at 250 and 250 mg/L).Sodium nitrate was used as the nitrogen source.Three runs were performed over two weeks.In each run, four bottles were assigned to the control (no NT, no MCFA) to obtain a robust average value, three bottles for the NT and blank (no substrate), and two bottles for each of the ten treatments (NT + MCFA and NT + MCFA/MCFA).The average values of the bottles for each treatment in each run were used as experimental replicates.
Rumen content samples were immediately transported to the laboratory in thermal flasks.The interval between sampling and the next sample processing was 20-30 min.In the laboratory, rumen content was strained The experimental substrates consisted of (on a DM basis) corn silage (300 g/kg), alfalfa silage (300 g/kg), and barley (400 g/kg).The dried feeds were ground and passed through a 1-mm screen.The chemical composition per kilogram of substrate (DM basis) was as follows: organic matter, 951 g/kg; crude protein, 154 g/kg; ether extract, 25 g/kg; starch, 306 g/kg; neutral detergent fiber (NDF), 354 g/kg; and acid detergent fiber (ADF), 193 g/kg.

In vitro incubations
The in vitro incubations were conducted over 24 h in 120-mL serum bottles.The dried ground substrate (200 mg) was weighed into sterile CO 2 -flushed serum bottles the day before incubation.The culture fluid (20 mL) was dispensed into each serum bottle using a bottle-top dispenser (Calibrex 530 Salutae, Socorex, Switzerland) under a stream of CO 2 .The culture was prepared by mixing composite rumen fluid with a medium (1:3, v/v) as described previously 75 .The resulting mixture was immediately gassed with CO 2 at 39 °C for 10 min before being added to the bottles.Sodium nitrate and MCFA were supplied to bottles by adding 200 µL of filter-sterilized distilled water and ethanol stock solutions, respectively, to reach the desired concentration in 20 mL of culture fluid.Equivalent amounts of water and ethanol were added to the control and blank bottles to compensate for the possible effects of solvents on fermentation.The initial headspace gas phase in all incubations was CO 2 .All serum bottles were sealed and placed in a temperature-controlled water bath (SW 22; Julabo, Germany) at 39 °C with a shaking frequency of 90 rpm for 24 h.

Sampling and chemical analyses
Total volume of gas produced was estimated from the headspace gas pressure using Boyle's law 76 .Headspace gas pressure was measured using a manometer (Traceable; Fisher Scientific, Pittsburgh, PA, USA) after 24 h of incubation.The headspace gas was then sampled by displacement into a tube (5 mL) prefilled with distilled water by inserting a 23 gauge needle through the stopper of the bottle.The CH 4 concentration in the headspace gas was measured using gas chromatography 77 .
The concentration of each VFA in the cultures was examined using gas chromatography 77 .VFA concentrations were calculated as the difference between concentrations in the fluid sample after 24 h of incubation and initial concentrations and were therefore reported as nVFA produced.The ammonia-N concentrations in the cultures were determined using the indophenol method 78 .The aDMd was determined gravimetrically by calculating the difference between the weight of the incubated substrate and the dry weight of the fermentation residue, normalized to the residue weight in the blank 77 .
The PCR reaction was performed with the following steps: initial denaturation at 95 °C for 45 s, annealing at 50 °C for 30 s, elongation at 68 °C for 30 s and a final 5 min extension at 68 °C; the reaction was put to hold at 4 °C.The number of cycles was adjusted separately for each sample to minimize chimeric sequence formation.The resulting PCR products were verified using agarose gel (1%) electrophoresis with staining of the gels with SYBR™ Green I Nucleic Acid Gel Stain (Thermo Fisher, USA).The banding patterns were documented using the Gel Doc XR + System (BioRad, USA).All PCRs were performed in triplicate and pooled.
We employed the 'vegan' package in R to visualize and assess community compositional differences (beta diversity) through NMDS (Fig. 3) analysis using the metaMDS function 81 .The ordination patterns were acceptable, as the stress values of the two-dimensional NMDS analysis were below 0.20 82 (stress = 0.1686036).NMDS stress values are reported after 100 tries and the best solution was repeated 13 times.Next, we utilized ANCOM-BC (version 2.2.2), implemented through the ' ANCOMBC' package 83 , to identify variations in taxa abundance between the groups at the family level (Fig. 4).We applied the Holm-Bonferroni method to adjust for multiple testing and to decrease the likelihood of type I error 84 .Nonsignificant results (P > 0.05) were replaced by 0.

Statistical analysis
The main effect of treatment was analyzed using the PROC MIXED procedure in SAS (SAS Enterprise Guide 6.1, SAS Institute Inc., Cary, NC, USA) according to a randomized complete block design.Runs (n = 3) were the blocking factors.Prior to the statistical analysis, technical replicates (parallel bottles for each treatment) were averaged per run.The model was: where Y ij is the dependent variable, µ is the overall mean, T i is the fixed effect of the treatment (i = 12 levels; control, nitrate, and 10 combinations of nitrate with MCFA), R j is the random effect of the run (j = 1, 2, and 3), and e ij is the residual error.Treatment means were compared with the control using Dunnett's adjustment, and differences between each treatment and the control were considered significant at P < 0.05.

Table 2 .
Impacts of MCFA + NT on in vitro volatile fatty acids production and proportion.† Net production of volatile fatty acids; ‡ acetate:propionate.*Means within a column differ significantly (P < 0.05) from the corresponding control (0 mg/L).SEM, standard error of the mean.

Table 3 .
The influence of the NT + MCFA treatments on α-diversity of microbial communities in vitro.*Means within a column differ significantly (P < 0.05) from the corresponding control (0 mg/L).SEM, standard error of the mean.